The authors would like to thank Shawna Rideout-Gros and Alexandra Garces for their help with the animal care, and Ping Wu for her technical assistance. Support for this work was provided by the National Institutes of Health under grant no. EB003268, and the Canada Research Chairs Program.

1. Ultrasound and MRI Setup
An MRI-compatible three-axis focused ultrasound system was used in this study (FUS Instruments, Inc., Toronto, Ontario, Canada). Two different ultrasound transducers were used: an in-house constructed 551.5 kHz spherically-focused transducer (radius of curvature = 60 mm, external diameter = 75 mm, internal diameter = 20 mm), and a 1.503 MHz, 8-sector array with integrated PZT hydrophone (Imasonic Inc., Voray-sur-L'Orgnon, France) driven as a single element spherically focused transducer (FN = 0.8, aperture = 10 cm). An MRI-compatible PVDF receiver8 was used to record acoustic emissions when the 551.5 kHz transducer was used. If different equipment is used, the following are suggested:
<ol>
	<li>Select an appropriate, calibrated ultrasound transducer with a center frequency in the range 0.25 MHz -1.5 MHz and an f-number (radius of curvature/aperture) of 1 or less.</li>
	<li>Place a water bath filled with warm, degassed, de-ionized water on the bed of a 1.5 T or 3 T MRI. The water bath should have a top-plate which can hold the animals. Mount the ultrasound transducer in the tank on an MRI compatible three-axis positioning stage or system9, facing the water surface.</li>
	<li>Run the transducer cable to the ultrasound driving equipment through a penetration panel.</li>
	<li>Generate the transducer driving signal using a function generator and power amplifier. Use an external matching circuit to minimize the reflected electrical power.</li>
	<li>Knowing the transducer focal length, place the transducer focus at the water surface and sonicate the water surface to generate a visible water fountain. Steps 1.6 and 1.7 describe how to register the focus using the generated water fountain. A more precise method for locating the focus, which involves sonicating a heat absorbing gel and imaging the resulting temperature elevation at the focus with MRI, can be found in9.</li>
	<li>Mark the location of the focus using a marker that will be visible on the MR images. This can be done using a plate with a center hole that will fill with water when placed over the focus. The water-filled hole will be visible on the MRI and will provide focal coordinates in two planes, while the coordinate in the axial direction of the transducer can be determined from the water surface.</li>
	<li>Using a 3-plane localizer sequence, image the focus marker and record the location of the focus in the MRI coordinate system.</li>
	<li>To monitor acoustic emissions, mount an MR-compatible hydrophone8 that is used as a passive cavitation detector (PCD) in the water bath directed at the transducer focus, or use a transducer with integrated hydrophone.</li>
	<li>Run the PCD cables to a scope card that is sufficiently fast to capture bursts of up to 10 ms at PRFs up to 2 Hz (e.g. ATS460, AlazarTech, Pointe-Claire, Quebec, Canada). Cables must be grounded to the penetration panel or RF shield to minimize noise.</li>
</ol>
2. Animal Preparation
<ol>
	<li>Anesthetize the animals using isofluorane gas. Since isofluorane has an effect on BBB disruption10, the animals should be taken off the gas 10 minutes prior to the start of the experiment. Place ocular lubricant ointment in each eye at the start of anesthesia to protect the cornea from desiccation or other injury.</li>
	<li>Using an electric razor shave the fur from the top of the animal's head and neck, then remove the remaining fur using a depilatory cream (e.g Nair, Church &amp; Dwight Co., Princeton, NJ, USA) and rinse the scalp with mild soap and water.</li>
	<li>Prepare the tail with a betadine skin scrub followed by a bridine wash. Perform a final wipedown with alcohol in order to visualize the tail vein.</li>
	<li>Insert a 22 gauge catheter with 3-way stopcock into the tail vein and flush with a heparin/saline mixture (33 U/mL) to prevent clot formation in the catheter.</li>
	<li>Deliver injectable anesthetics (40-50 mg/kg ketamine, 10 mg/kg xylazine) via intramuscular injection, and remove the animal from the isofluorane.</li>
	<li>Place the anesthetized animal supine on the ultrasound positioning system with the top of the head contacting the waterbath through a hole in the top-plate (Fig.1). A small amount of ultrasound gel maybe applied to the top of the animal's head to minimize the chance of trapped airbubbles.</li>
	<li>Tape the legs to the positioning system. The head may be held in place using either a bite bar, if available, or tape placed firmly across the chin.</li>
	<li>Cover the animal with a towel and circulating water blanket in order to keep it warm.</li>
</ol>
3. Target Selection
<ol>
	<li> Acquire baseline axial T2-weighted and T1-weighted MR images of the brain. If using a 1.5 T MRI and dedicated head-sized RF-receive surface coil, suitable scan parameters might be: 
	 
	T2 weighted: FSE, TE = 60 ms, TR =2000 ms 
	T1 weighted: FSE, TE=10 ms, TR=500 ms </li>
	<li>Select the target from the T2-weighted scans, avoiding the ventricles and the brain midline, and selecting a mid-brain depth.</li>
	<li>Move the transducer focus to the target location.</li>
</ol>
4. Microbubble Preparation
Definity microbubbles (Lantheus Medical Imaging, MA, USA) are used by several groups for microbubble mediated FUS induced BBBD2,5,7. Appropriate dosages for other microbubble types can be found in the literature11,12.
<ol>
	<li>Activate the Definity microbubbles and slowly draw up a small volume into a 1 mL syringe using an 18-gauge needle.</li>
	<li>Remove trapped air from the syringe by gently moving the plunger back and forth. Do not tap the syringe as this can break the bubbles.</li>
	<li>Dilute the Definity in normal saline in a ratio of 10:1 saline to Definty by slowly injecting the required volume of Definity into a syringe of saline. If an infusion delivery is used, the Definity can be further diluted, to 50:1 or 100:1.</li>
	<li>Gently invert the syringe to thoroughly mix the Definity and saline until an even appearance is achieved. Gentle inversion of the syringe immediately prior to injection may also be necessary if the bubbles have started to float out of suspension.</li>
	<li>Calculate the required dose volume based on 0.02 mL/kg of Definity, or 0.2 mL/kg of the solution (at 10:1 dilution).</li>
</ol>
5. Ultrasound Delivery
<ol>
	<li>Set the sonication parameters, using low duty cycle bursts, not continuous wave sonications. Appropriate sonication parameters at 0.5 MHz are 0.23 MPa in situ pressure, 10 ms bursts at 1 Hz PRF, 2 min. total sonication time. At 1.5 MHz appropriate in situ pressures fall around 0.45-0.5 MPa.</li>
</ol>
Appropriate pressures at different frequencies can be estimated using a mechanical index of 0.46 13.
<ol start="2">
	<li>Check that the animal's head is still coupled to the water.</li>
	<li>Inject the microbubbles slowly into the tail vein catheter and flush with 0.5 mL normal saline. Begin the sonication simultaneously with the start of injection.</li>
</ol>
6. MRI Evaluation of Treatment Outcome
<ol>
	<li>Following the sonication, inject an MRI contrast agent (e.g. 0.2 ml/kg Omniscan, GE Healthcare) via the tail vein catheter followed by a 0.5 mL saline flush.</li>
	<li>Perform T1-weighted imaging until the contrast peak has passed. Sonication sites which have been successfully disrupted will show greater enhancement than the surrounding tissue.</li>
	<li>Perform T2-weighted imaging to check for edema. High signal at the sonication sites is indicative of edema.</li>
</ol>
7. Representative Results
MRI contrast agents can be successfully delivered through the BBB using focused ultrasound and circulating microbubbles. Figure 2 shows typical pre and post-FUS T1-w images. Figure 2B shows a contrast enhanced (CE) T1-w image with distinct focal openings in four sonicated locations. Sonication locations 1 and 2 show particularly bright enhancement. In Fig.3 locations 1 and 2 can also be seen to correspond with T2-w high signal, indicating edema.
The extent of T2-w edema can sometimes be more easily visualized on sagittal slices. Figure 4 shows CE-T1-w and T2-w sagittal slices through sonication locations 1 and 3. Edema is visible at location 1 but not location 3.
Spectral analysis of captured acoustic emissions data (Fig.5) may show harmonic emissions and/or sub/ultra harmonic emissions when stable cavitation is occurring. Harmonics can also arise from tissue non-linearities, while sub and ultraharmonic emissions can only occur as a result of bubble activity14. At higher pressures wideband emissions indicating inertial cavitation may also be detected. However these have been associated with greater amounts of red blood cell extravasations and microdamage than sonications without inertial cavitation11.
The use of higher sonication frequencies results in more localized openings due to the smaller focal spot size. Figure 6 shows that higher frequencies can be used to create smaller regions of opening. This allows investigation of effects mid-brain with fewer near-skull effects.
<img alt="Figure 1." src="/files/ftp_upload/3555/3555fig1.jpg" /> 
Figure 1. Experimental setup.
<img alt="Figure 2." src="/files/ftp_upload/3555/3555fig2.jpg" /> 
Figure 2. Pre (left) and post (right) treatment T1-w images of a rat brain showing enhancement at four sonication locations.
<img alt="Figure 3." src="/files/ftp_upload/3555/3555fig3.jpg" /> 
Figure 3. Pre (left) and post (right) treatment T2-w images of a rat brain (same animal as Fig.2) showing T2-w edema at sonication locations 1 and 2.
<img alt="Figure 4." src="/files/ftp_upload/3555/3555fig4.jpg" /> 
Figure 4. Post treatment sagittal T1-w (left) and T2-w (right) images from the same rat brain as Figs. 2 and 3. The opening at location 1 (left) correlates with T2-w edema (right). Location 3 shows opening (left) but no T2-w edema.
<img alt="Figure 5." src="/files/ftp_upload/3555/3555fig5.jpg" /> 
Figure 5. Frequency spectrum from data captured during a single 10 ms burst at 551.5 kHz. The fundamental frequency ( f0) as well as harmonics (2f0) and sub/ultraharmonics (0.5 f0, 1.5f0) are visible.
<img alt="Figure 6." src="/files/ftp_upload/3555/3555fig6.jpg" /> 
Figure 6. Post treatment CE-T1-w axial (left) and sagittal (right) images from a rat brain sonicated in four locations at 1.503 MHz. BBB openings at this frequency are seen to be more localized.

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	<li>Kinoshita, M., McDannold, N., Jolesz, F. A., Hynynen, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noninvasive+localized+delivery+of+Herceptin+to+the+mouse+brain+by+MRI-guided+focused+ultrasound-induced+blood-brain+barrier+disruption.">Noninvasive localized delivery of Herceptin to the mouse brain by MRI-guided focused ultrasound-induced blood-brain barrier disruption.</a> Proc. Natl. Acad. Sci. U.S.A. 103, 11719-11723 (2006).</li><li>Jord&#227;o, J. F., Ayala-Grosso, C. 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Am. 126, 3344-3349 (2009).</li><li>McDannold, N., Vykhodtseva, N., Hynynen, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood-brain+barrier+disruption+induced+by+focused+ultrasound+and+circulating+preformed+microbubbles+appears+to+be+characterized+by+the+mechanical+index.">Blood-brain barrier disruption induced by focused ultrasound and circulating preformed microbubbles appears to be characterized by the mechanical index.</a> Ultrasound Med. Biol. 34, 834-840 (2008).</li><li>Neppiras, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acoustic+Cavitation.">Acoustic Cavitation.</a> Physics Reports (Review Section of Physics Letters). 61, 159-251 (1980).</li><li>O&#39;Reilly, M. 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K. Hynynen and R. Chopra are co-founders of FUS Instruments, Inc. R. Chopra, A. Waspe and K. Hynynen are shareholders in FUS Instruments, Inc. K. Hynynen receives research support from FUS instruments, Inc.

Preparation of the animals and microbubbles are the most critical aspects of this procedure. The hair on the animal's head must be entirely removed to avoid attenuating the ultrasound beam. The BBB can be disrupted under isofluorane anesthetic, however, it becomes more difficult to achieve consistent opening.
The microbubbles should always be handled with care and small gauge, large diameter needles must be used when drawing up, in order to avoid breaking them. Similarly, the smallest gauge c...

<table border="1">
	<tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 </tr>
 <tr>
 <td>Small Animal Focused Ultrasound System</td>
 <td>FUS Instruments, Inc.</td>
 <td>RK-100</td>
 </tr>
 <tr>
 <td>Definity</td>
 <td>Lantheus Medical Imaging</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Omniscan</td>
 <td>GE Healthcare</td>
 <td>&nbsp;</td>
 </tr>
	 </tbody>
 </table>

mri-guided disruption of the blood-brain barrier using transcranial focused ultrasound in a rat model

Focused ultrasound (FUS) disruption of the blood-brain barrier (BBB) is an increasingly investigated technique for circumventing the BBB1-5. The BBB is a significant obstacle to pharmaceutical treatments of brain disorders as it limits the passage of molecules from the vasculature into the brain tissue to molecules less than approximately 500 Da in size6. FUS induced BBB disruption (BBBD) is temporary and reversible4 and has an advantage over chemical means of inducing BBBD by being highly localized. FUS induced BBBD provides a means for investigating the effects of a wide range of therapeutic agents on the brain, which would not otherwise be deliverable to the tissue in sufficient concentration. While a wide range of ultrasound parameters have proven successful at disrupting the BBB2,5,7, there are several critical steps in the experimental procedure to ensure successful disruption with accurate targeting. This protocol outlines how to achieve MRI-guided FUS induced BBBD in a rat model, with a focus on the critical animal preparation and microbubble handling steps of the experiment.

Watch this Scientific Journal Video about MRI-guided Disruption of the Blood-brain Barrier using Transcranial Focused Ultrasound in a Rat Model at JoVE.com

MRI-guided Disruption of the Blood-brain Barrier using Transcranial Focused Ultrasound in a Rat Model

Microbubble-mediated focused ultrasound disruption of the blood-brain barrier (BBB) is a promising technique for non-invasive targeted drug delivery in the brain1-3. This protocol outlines the experimental procedure for MRI-guided transcranial BBB disruption in a rat model.

Focused ultrasound (FUS) disruption of the blood-brain barrier (BBB) is an increasingly investigated technique for circumventing the BBB1-5. The BBB is a ...

mri-guided-disruption-blood-brain-barrier-using-transcranial-focused

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17.8K Views.  Sunnybrook Research Institute. Микропузырьков опосредованного сфокусированного ультразвука нарушение гематоэнцефалического барьера (ГЭБ) является перспективной техникой для неинвазивного целевой доставки лекарств в мозг 1-3. Этот протокол описывает экспериментальные процедур...

Video: MRI-guided Disruption of the Blood-brain Barrier using Transcranial Focused Ultrasound in a Rat Model

Multi-modal Imaging of Angiogenesis in a Nude Rat Model of Breast Cancer Bone Metastasis Using Magnetic Resonance Imaging, Volumetric Computed Tomography and Ultrasound

Angiogenesis is an essential feature of cancer growth and metastasis formation. In bone metastasis, angiogenic factors are pivotal for tumor cell proliferation in the bone marrow cavity as well as for interaction of tumor and bone cells resulting in local bone destruction. Our aim was to develop a model of experimental bone metastasis that allows in vivo assessment of angiogenesis in skeletal lesions using non-invasive imaging techniques.
For this purpose, we injected 105 MDA-MB-231 human breast cancer cells into the superficial epigastric artery, which precludes the growth of metastases in body areas other than the respective hind leg1. Following 25-30 days after tumor cell inoculation, site-specific bone metastases develop, restricted to the distal femur, proximal tibia and proximal fibula1. Morphological and functional aspects of angiogenesis can be investigated longitudinally in bone metastases using magnetic resonance imaging (MRI), volumetric computed tomography (VCT) and ultrasound (US).
MRI displays morphologic information on the soft tissue part of bone metastases that is initially confined to the bone marrow cavity and subsequently exceeds cortical bone while progressing. Using dynamic contrast-enhanced MRI (DCE-MRI) functional data including regional blood volume, perfusion and vessel permeability can be obtained and quantified2-4. Bone destruction is captured in high resolution using morphological VCT imaging. Complementary to MRI findings, osteolytic lesions can be located adjacent to sites of intramedullary tumor growth. After contrast agent application, VCT angiography reveals the macrovessel architecture in bone metastases in high resolution, and DCE-VCT enables insight in the microcirculation of these lesions5,6. US is applicable to assess morphological and functional features from skeletal lesions due to local osteolysis of cortical bone. Using B-mode and Doppler techniques, structure and perfusion of the soft tissue metastases can be evaluated, respectively. DCE-US allows for real-time imaging of vascularization in bone metastases after injection of microbubbles7.
In conclusion, in a model of site-specific breast cancer bone metastases multi-modal imaging techniques including MRI, VCT and US offer complementary information on morphology and functional parameters of angiogenesis in these skeletal lesions.

In the pathogenesis of bone metastasis, angiogenesis is a crucial process and therefore represents a target for imaging and therapy. Here, we present a rat model of site-specific breast cancer bone metastasis and describe strategies to non-invasively image angiogenesis in vivo using magnetic resonance imaging, volumetric computed tomography and ultrasound.

Angiogenesis is an essential feature of cancer growth and metastasis formation. In bone metastasis, angiogenic factors are pivotal for tumor cell ...

Rat Model of Blood-brain Barrier Disruption to Allow Targeted Neurovascular Therapeutics

Endothelial cells with tight junctions along with the basement membrane and astrocyte end feet surround cerebral blood vessels to form the blood-brain barrier1. The barrier selectively excludes molecules from crossing between the blood and the brain based upon their size and charge. This function can impede the delivery of therapeutics for neurological disorders. A number of chemotherapeutic drugs, for example, will not effectively cross the blood-brain barrier to reach tumor cells2. Thus, improving the delivery of drugs across the blood-brain barrier is an area of interest.
The most prevalent methods for enhancing the delivery of drugs to the brain are direct cerebral infusion and blood-brain barrier disruption3. Direct intracerebral infusion guarantees that therapies reach the brain; however, this method has a limited ability to disperse the drug4. Blood-brain barrier disruption (BBBD) allows drugs to flow directly from the circulatory system into the brain and thus more effectively reach dispersed tumor cells. Three methods of barrier disruption include osmotic barrier disruption, pharmacological barrier disruption, and focused ultrasound with microbubbles. Osmotic disruption, pioneered by Neuwelt, uses a hypertonic solution of 25% mannitol that dehydrates the cells of the blood-brain barrier causing them to shrink and disrupt their tight junctions. Barrier disruption can also be accomplished pharmacologically with vasoactive compounds such as histamine5 and bradykinin6. This method, however, is selective primarily for the brain-tumor barrier7. Additionally, RMP-7, an analog of the peptide bradykinin, was found to be inferior when compared head-to-head with osmotic BBBD with 25% mannitol8. Another method, focused ultrasound (FUS) in conjunction with microbubble ultrasound contrast agents, has also been shown to reversibly open the blood-brain barrier9. In comparison to FUS, though, 25% mannitol has a longer history of safety in human patients that makes it a proven tool for translational research10-12.
In order to accomplish BBBD, mannitol must be delivered at a high rate directly into the brain's arterial circulation. In humans, an endovascular catheter is guided to the brain where rapid, direct flow can be accomplished. This protocol models human BBBD as closely as possible. Following a cut-down to the bifurcation of the common carotid artery, a catheter is inserted retrograde into the ECA and used to deliver mannitol directly into the internal carotid artery (ICA) circulation. Propofol and N2O anesthesia are used for their ability to maximize the effectiveness of barrier disruption13. If executed properly, this procedure has the ability to safely, effectively, and reversibly open the blood-brain barrier and improve the delivery of drugs that do not ordinarily reach the brain 8,13,14.

Blood-brain barrier disruption aids the delivery of certain drugs to the brain. Mannitol delivered intra-arterially shrinks cells surrounding blood vessels in order to physically disrupt the barrier.

Endothelial cells with tight junctions along with the basement membrane and astrocyte end feet surround cerebral blood vessels to form the blood-brain ...

A Model of Chronic Nutrient Infusion in the Rat

Chronic exposure to excessive levels of nutrients is postulated to affect the function of several organs and tissues and to contribute to the development of the many complications associated with obesity and the metabolic syndrome, including type 2 diabetes. To study the mechanisms by which excessive levels of glucose and fatty acids affect the pancreatic beta-cell and the secretion of insulin, we have established a chronic nutrient infusion model in the rat. The procedure consists of catheterizing the right jugular vein and left carotid artery under general anesthesia; allowing a 7-day recuperation period; connecting the catheters to the pumps using a swivel and counterweight system that enables the animal to move freely in the cage; and infusing glucose and/or Intralipid (a soybean oil emulsion which generates a mixture of approximately 80% unsaturated/20% saturated fatty acids when infused with heparin) for 72 hr. This model offers several advantages, including the possibility to finely modulate the target levels of circulating glucose and fatty acids; the option to co-infuse pharmacological compounds; and the relatively short time frame as opposed to dietary models. It can be used to examine the mechanisms of nutrient-induced dysfunction in a variety of organs and to test the effectiveness of drugs in this context.

A protocol for chronic infusions of glucose and Intralipid in rats is described. This model can be used to study the impact of nutrient excess on organ function and physiological parameters.

Chronic exposure to excessive levels of nutrients is postulated to  affect the function of several organs and tissues and to contribute to the  ...

Stretch in Brain Microvascular Endothelial Cells (cEND) as an In Vitro Traumatic Brain Injury Model of the Blood Brain Barrier

Due to the high mortality incident brought about by traumatic brain injury (TBI), methods that would enable one to better understand the underlying mechanisms involved in it are useful for treatment. There are both in vivo and in vitro methods available for this purpose. In vivo models can mimic actual head injury as it occurs during TBI. However, in vivo techniques may not be exploited for studies at the cell physiology level. Hence, in vitro methods are more advantageous for this purpose since they provide easier access to the cells and the extracellular environment for manipulation.
Our protocol presents an in vitro model of TBI using stretch injury in brain microvascular endothelial cells. It utilizes pressure applied to the cells cultured in flexible-bottomed wells. The pressure applied may easily be controlled and can produce injury that ranges from low to severe. The murine brain microvascular endothelial cells (cEND) generated in our laboratory is a well-suited model for the blood brain barrier (BBB) thus providing an advantage to other systems that employ a similar technique. In addition, due to the simplicity of the method, experimental set-ups are easily duplicated. Thus, this model can be used in studying the cellular and molecular mechanisms involved in TBI at the BBB.

Stretch in Brain Microvascular Endothelial Cells cEND as an In Vitro Traumatic Brain Injury Model of the Blood Brain Barrier

In vitro traumatic brain injury models are being developed to reproduce in vivo brain deformation. Stretch-induced injury has been employed for astrocytes, neurons, glial cells, aortic, and brain endothelial cells. However, our system uses a blood brain barrier (BBB) model that possesses properties constituting a legitimate model of the BBB to establish an in vitro&#160;TBI model.

Due to the high mortality incident brought about by traumatic brain injury (TBI), methods that would enable one to better understand the underlying ...

Setting-up an In Vitro Model of Rat Blood-brain Barrier (BBB): A Focus on BBB Impermeability and Receptor-mediated Transport

The blood brain barrier (BBB) specifically regulates molecular and cellular flux between the blood and the nervous tissue. Our aim was to develop and characterize a highly reproducible rat syngeneic in vitro model of the BBB using co-cultures of primary rat brain endothelial cells (RBEC) and astrocytes to study receptors involved in transcytosis across the endothelial cell monolayer. Astrocytes were isolated by mechanical dissection following trypsin digestion and were frozen for later co-culture. RBEC were isolated from 5-week-old rat cortices. The brains were cleaned of meninges and white matter, and mechanically dissociated following enzymatic digestion. Thereafter, the tissue homogenate was centrifuged in bovine serum albumin to separate vessel fragments from nervous tissue. The vessel fragments underwent a second enzymatic digestion to free endothelial cells from their extracellular matrix. The remaining contaminating cells such as pericytes were further eliminated by plating the microvessel fragments in puromycin-containing medium. They were then passaged onto filters for co-culture with astrocytes grown on the bottom of the wells. RBEC expressed high levels of tight junction (TJ) proteins such as occludin, claudin-5 and ZO-1 with a typical localization at the cell borders. The transendothelial electrical resistance (TEER) of brain endothelial monolayers, indicating the tightness of TJs reached 300 ohm&#183;cm2 on average. The endothelial permeability coefficients (Pe) for lucifer yellow (LY) was highly reproducible with an average of 0.26 &#177;&#160;0.11 x 10-3 cm/min. Brain endothelial cells organized in monolayers expressed the efflux transporter P-glycoprotein (P-gp), showed a polarized transport of rhodamine 123, a ligand for P-gp, and showed specific transport of transferrin-Cy3 and DiILDL across the endothelial cell monolayer. In conclusion, we provide a protocol for setting up an in vitro BBB model that is highly reproducible due to the quality assurance methods, and that is suitable for research on BBB transporters and receptors.

Setting-up an In Vitro Model of Rat Blood-brain Barrier BBB: A Focus on BBB Impermeability and Receptor-mediated Transport

The aim of the present study was to validate the reproducibility of an in vitro BBB model involving a rat syngeneic co-culture of endothelial cells and astrocytes. The endothelial cell monolayer presented high TEER and low LY permeability. Expression of specific TJ proteins, functional responses to inflammation and functionality of transporters and receptors were assessed.

The blood brain barrier (BBB) specifically regulates molecular and cellular flux between the blood and the nervous tissue. Our aim was to develop and ...

Measuring Ascending Aortic Stiffness In Vivo in Mice Using Ultrasound

We present a protocol for measuring in vivo aortic stiffness in mice using high-resolution ultrasound imaging. Aortic diameter is measured by ultrasound and aortic blood pressure is measured invasively with a solid-state pressure catheter. Blood pressure is raised then lowered incrementally by intravenous infusion of vasoactive drugs phenylephrine and sodium nitroprusside. Aortic diameter is measured for each pressure step to characterize the pressure-diameter relationship of the ascending aorta. Stiffness indices derived from the pressure-diameter relationship can be calculated from the data collected. Calculation of arterial compliance is described in this protocol.
This technique can be used to investigate mechanisms underlying increased aortic stiffness associated with cardiovascular disease and aging. The technique produces a physiologically relevant measure of stiffness compared to ex vivo approaches because physiological influences on aortic stiffness are incorporated in the measurement. The primary limitation of this technique is the measurement error introduced from the movement of the aorta during the cardiac cycle. This motion can be compensated by adjusting the location of the probe with the aortic movement as well as making multiple measurements of the aortic pressure-diameter relationship and expanding the experimental group size.

Measuring Ascending Aortic Stiffness In Vivo in Mice Using Ultrasound

We describe a technique for measuring aortic stiffness from its pressure-diameter relationship in vivo in mice. Aortic diameter is recorded by ultrasound and aortic pressure is measured invasively with a solid-state pressure catheter. Blood pressure is changed incrementally and the resulting diameter is measured.

We present a protocol for measuring in vivo aortic stiffness in mice using high-resolution ultrasound imaging. Aortic diameter is measured by ultrasound ...

Oxygen-Glucose Deprivation and Reoxygenation as an In Vitro Ischemia-Reperfusion Injury Model for Studying Blood-Brain Barrier Dysfunction

Ischemia-Reperfusion (IR) injury is known to contribute significantly to the morbidity and mortality associated with ischemic strokes. Ischemic cerebrovascular accidents account for 80% of all strokes. A common cause of IR injury is the rapid inflow of fluids following an acute/chronic occlusion of blood, nutrients, oxygen to the tissue triggering the formation of free radicals.
Ischemic stroke is followed by blood-brain barrier (BBB) dysfunction and vasogenic brain edema. Structurally, tight junctions (TJs) between the endothelial cells play an important role in maintaining the integrity of the blood-brain barrier (BBB). IR injury is an early secondary injury leading to a non-specific, inflammatory response. Oxidative and metabolic stress following inflammation triggers secondary brain damage including BBB permeability and disruption of tight junction (TJ) integrity.
Our protocol presents an in vitro example of oxygen-glucose deprivation and reoxygenation (OGD-R) on rat brain endothelial cell TJ integrity and stress fiber formation. Currently, several experimental in vivo models are used to study the effects of IR injury; however they have several limitations, such as the technical challenges in performing surgeries, gene dependent molecular influences and difficulty in studying mechanistic relationships. However, in vitro models may aid in overcoming many of those limitations. The presented protocol can be used to study the various molecular mechanisms and mechanistic relationships to provide potential therapeutic strategies. However, the results of in vitro studies may differ from standard in vivo studies and should be interpreted with caution.

Oxygen-Glucose Deprivation and Reoxygenation as an In Vitro Ischemia-Reperfusion Injury Model for Studying Blood-Brain Barrier Dysfunction

Ischemia-Reperfusion (IR) injury is associated with a high rate of morbidity and mortality. The goal of the in vitro model of oxygen-glucose deprivation and reoxygenation (OGD-R) described here is to assess the effects of ischemia reperfusion injury on a variety of cells, particularly in blood-brain barrier (BBB) endothelial cells.

Ischemia-Reperfusion (IR) injury is known to contribute significantly to the morbidity and mortality associated with ischemic strokes. Ischemic ...

MRI-guided dmPFC-rTMS as a Treatment for Treatment-resistant Major Depressive Disorder

Here we outline the protocol for magnetic resonance imaging (MRI) guided repetitive transcranial magnetic stimulation (rTMS) to the dorsal medial prefrontal cortex (dmPFC) in patients with major depressive disorder (MDD). Technicians used a neuronavigation system to process patient MRIs to generate a 3-dimensional head model. The head model was subsequently used to identify patient-specific stimulatory targets. The dmPFC was stimulated daily for 20 sessions. Stimulation intensity was titrated to address scalp pain associated with rTMS. Weekly assessments were conducted on the patients using the Hamilton Rating Scale for Depression (HamD17) and Beck Depression Index II (BDI-II). Treatment-resistant MDD patients achieved significant improvements on both HAMD and BDI-II. Of note, angled, double-cone coil rTMS at 120% resting motor threshold allows for optimal stimulation of deeper midline prefrontal regions, which results in a possible therapeutic application for MDD. One major limitation of the rTMS field is the heterogeneity of treatment parameters across studies, including duty cycle, number of pulses per session and intensity. Further work should be done to clarify the effect of stimulation parameters on outcome. Future dmPFC-rTMS work should include sham-controlled studies to confirm its clinical efficacy in MDD.

Here we outline the procedure for MRI-guided repetitive transcranial magnetic stimulation to the dorsomedial prefrontal cortex as an experimental treatment for major depressive disorder.

Here we outline the protocol for magnetic resonance imaging (MRI) guided repetitive transcranial magnetic stimulation (rTMS) to the dorsal medial ...

In Vitro Assays to Assess Blood-brain Barrier Mesh-like Vessel Formation and Disruption

Blood-brain barrier (BBB) coverage plays a central role in the homeostasis of the central nervous system (CNS). The BBB is dynamically maintained by astrocytes, pericytes and brain endothelial cells (BECs). Here, we detail methods to assess BBB coverage using single cultures of immortalized human BECs, single cultures of primary mouse BECs, and a humanized triple culture model (BECs, astrocytes and pericytes) of the BBB. To highlight the applicability of the assays to disease states, we describe the effect of oligomeric amyloid-&#946; (oA&#946;), which is an important contributor to Alzheimer&#39;s disease (AD) progression, on BBB coverage. Further, we utilize the epidermal growth factor (EGF) to illuminate the drug screening potential of the techniques. Our results show that single and triple cultured BECs form meshwork-like structures under basal conditions, and that oA&#946; disrupts this cell meshwork formation and degenerates the preformed mesh structures, but EGF blocks this disruption. Thus, the techniques described are important for dissecting fundamental and disease-relevant processes that modulate BBB coverage.

In Vitro Assays to Assess Blood-brain Barrier Mesh-like Vessel Formation and Disruption

Maintaining blood-brain barrier coverage is key for the homeostasis of the central nervous system. This protocol describes in vitro techniques to delineate the fundamental and pathological processes that modulate blood-brain barrier coverage.

Blood-brain barrier (BBB) coverage plays a central role in the homeostasis of the central nervous system (CNS). The BBB is dynamically maintained by ...

Improved Method for the Establishment of an In Vitro Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells

The aim of this protocol presents an optimized procedure for the purification and cultivation of pBECs and to establish in vitro blood-brain barrier (BBB) models based on pBECs in mono-culture (MC), MC with astrocyte-conditioned medium (ACM), and non-contact co-culture (NCC) with astrocytes of porcine or rat origin. pBECs were isolated and cultured from fragments of capillaries from the brain cortices of domestic pigs 5-6 months old. These fragments were purified by careful removal of meninges, isolation and homogenization of grey matter, filtration, enzymatic digestion, and centrifugation. To further eliminate contaminating cells, the capillary fragments were cultured with puromycin-containing medium. When 60-95% confluent, pBECs growing from the capillary fragments were passaged to permeable membrane filter inserts and established in the models. To increase barrier tightness and BBB characteristic phenotype of pBECs, the cells were treated with the following differentiation factors: membrane permeant 8-CPT-cAMP (here abbreviated cAMP), hydrocortisone, and a phosphodiesterase inhibitor, RO-20-1724 (RO). The procedure was carried out over a period of 9-11 days, and when establishing the NCC model, the astrocytes were cultured 2-8 weeks in advance. Adherence to the described procedures in the protocol has allowed the establishment of endothelial layers with highly restricted paracellular permeability, with the NCC model showing an average transendothelial electrical resistance (TEER) of 1249 &#177; 80 &#937; cm2, and paracellular permeability (Papp) for Lucifer Yellow of 0.90 10-6 &#177; 0.13 10-6 cm sec-1 (mean &#177; SEM, n=55). Further evaluation of this pBEC phenotype showed good expression of the tight junctional proteins claudin 5, ZO-1, occludin and adherens junction protein p120 catenin. The model presented can be used for a range of studies of the BBB in health and disease and, with the highly restrictive paracellular permeability, this model is suitable for studies of transport and intracellular trafficking.

Improved Method for the Establishment of an In Vitro Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells

The aim of the protocol is to present an optimized procedure for the establishment of an in vitro blood-brain barrier (BBB) model based on primary porcine brain endothelial cells (pBECs). The model shows high reproducibility, high tightness, and is suitable for studies of transport and intracellular trafficking in drug discovery.

The aim of this protocol presents an optimized procedure for the purification and cultivation of pBECs and to establish in vitro blood-brain barrier (BBB) ...